2.1. Chemicals and drugs
MPTP was purchased from Solarbio (IM0290, Beijing, China) and stored at -20℃. MPP + was purchased from N137206 (ALADDIN, Shanghai, China) and stored at -20℃. Madopar tablet was purchased from Roche Pharmaceuticals (Shanghai, China) and stored at room temperature. Gp was presented by Hainan Wandailan Bio (Hainan, China) and stored at room temperature.
Drp1 antibody (#8570) was purchased from CST (Boston, USA). Mfn2 antibody (ab124773), OPA1 antibody (ab42364), TH antibody (ab112) were purchased from Abcam (MA, USA). DMEM high glucose was from Biological Industries Ltd (HaZafon, Israel). Penicillin-Streptomycin solution, 0.25% trypsin-EDTA solution without phenol red, NP-40 lysis buffer, MTT, BCA protein concentration determination kit, paraformaldehyde and dimethyl sulfoxide (DMSO), phosphate buffered saline (PBS, pH 7.4), Tween 20, 5×SDS-PAGE sample loading buffer and other reagents for cell culture and western blotting were obtained from Solarbio (Beijing, China).
2.2. Animals and treatments
Two month old C57BL/6 mice (20–25 g) were purchased from Huaxing experimental animal farm (Zhengzhou, China) and were housed in a room with ventilation system of the experimental animals. The room and cages temperature were automatically maintained at 20–24°C, and an average relative humidity was 45%-60% with a controlled 12h light/12h dark cycle everyday. All the animals had free access to feed and water. The experimental procedures were in accordance with the principles of laboratory animal welfare.
After two weeks of adaptive feeding, the mice were held in the rotarod test instrument to remove the individuals with movement problems. Then, seven experimental groups of mice (n = 10) were studied: (1) Control group: saline injection plus saline oral administration; (2) MPTP group: MPTP injection plus saline; (3) Gp-low dosage group (Gp-L): MPTP injection plus Garlic powder 13mg/kg/day; (4) Gp-middle dosage group (Gp-M): MPTP injection plus Gp 26mg/kg/day; (5) Gp-high dosage group (Gp-H): MPTP injection plus Gp 52mg/kg/day; (6) Positive Control group: MPTP injection plus Madopar 1.0mg/kg/day; (7) Control group with Gp: saline injection plus Gp 52mg/kg/day. In the first seven days, The Control group was intraperitoneally injected with saline while the experimental group was injected with MPTP (20 mg/kg). The experiment lasted for 30 days. The Control group was given saline and the experimental group was given corresponding drugs by oral administration.
2.3 N2a cell culture and treatment
The Neuro-2a (N2a) cells line (ATCC® CCL-131) were obtained from Cell Resource Center, Shanghai Institute of life sciences, Chinese Academy of Sciences (Shanghai, China). N2a cells were cultivated in Dulbecco’s modified eagle medium (DMEM, 1×, high glucose) Glutamax supplemented with 10% FBS, 100IU/ml of Penicillin and 100ug/ml of Streptomycin. Cells were maintained at 37℃ in a humidified incubator with 5% CO2 and 95% air. Cells were subcultured when 80–90% confluent and seeded at 1:4 ratio. Culture medium was renewed every 3 to 4 days.
MPP + was diluted in PBS at a concentration of 100mM, aliquoted and stored at − 20°C until used. The final concentration in complete medium was 2 mM[19]. Gp was diluted in complete medium at concentrations of 2.5mg/ml, 5mg/ml, 10mg/ml and was filtered by 0.22µm sterile filter element.
2.4 Animal behavior test
The rotarod test and Open-field instrument were purchased from RWD Life Technology (Shenzhen, China). The behavioral test was started 5 days before the end of administration. After all the behavior test, the mice were killed by corresponding treatment method for molecular biology experiment.
Rotarod test
The rotarod test was used to evaluate the balance and coordination ability of mice. The mice were put on the roller for three days before the test and trained for 3 minutes every day with the speed at 20 rpm. After training, the speed was set to 20 rpm for 20 seconds at first and accelerated to 30 rpm in 30 seconds. During the test, the residence time of the mice (Latency to falls) in the roller was recorded. The regular tests were 6 times, and then the average value was taken for Statistical analysis.
Open-field test
To assess locomotor and exploratory activity of PD mice, the open-field test was conducted on the 6th day after MPTP treatment. The open-field apparatus consisted of a square arena (40×40 cm) with 25cm-high opaque walls. The floor was divided into 16 equal squares by lines. The central area comprised the central 4 squares (20×20 cm). Each mouse was placed in the center of the apparatus and was observed. The number of crossing line and rearings made by the animal was recorded within 5 min. The apparatus was then cleaned with 75% alcohol and dried between trials. The experiment was repeated 3 times for each animal, and the average of 2 trials was calculated for statistical analyses.
Pole test
To determine the degree of bradykinesia and ability to movement balance of PD mice, the pole test was conducted on the 14th day after Gp treatment. In brief, animals were placed facing upward near the top of a wooden pole with a rough surface (10 mm in diameter and 55 cm in height). The time taken until they turned completely downward (defined as turn time, T-turn) and the time taken to arrive at the floor (locomotor activity time, T-LA) were recorded. The mice were pre-trained. Every mouse was tested for 3 times and the average of the three trials was calculated for statistical analyses.
2.5 Immunofluorescence histochemistry
After treatment and behavioral tests, 3 mice were sacrificed for immunohistochemistry to observe the protein expression of TH. The mice were transcardial perfused with precooling saline and 4% paraformaldehyde/PBS after anesthetization with 20% (w/v) uratan. The brains were removed rapidly and immersed in paraformaldehyde for 24 h at room temperature, and were settled by automatic tissue hydroextractor (Leica TP1020) and automatic embedding machine (Leica EG1150). Then, the paraffin block was sliced by Semi automatic rotary slicer (Leica RM2245). The thickness of tissue section was 4µm.
Follow the steps is tissue section in adhesive slides. The brain section in substantia nigra were dewaxed by dimethylbenzene, blocked by 3% H2O2 to inactivate endogenous peroxidase, and microwaved by thermal remediation with Sodium citrate antigen repair solution (C1032, Solarbio, Beijing, China). 5% BSA/PBS was used for antigen blocking incubation for 30 min. The sections were incubated overnight in 4℃ after the primary antibody TH (1:750) was added. The second day, the sections were then rinsed in PBS and incubated with donkey anti-rabbit IgG/Alex Fluor 488 (1: 250) at 37°C for 2h under dark conditions after rinsed in PBS and colored in DAPI staining solution (C0065, Solarbio, Beijing, China). After coating with anti fluorescence quenching sealing agent (P0128, beyotime, Shanghai, China), all stained sections were viewed and photographed under a microscope (ZEISS Axioscope 5, Germany). The fluorescence intensity was determined using Image-pro plus 6.0 software.
The process of N2a cells immunofluorescence was similar to tissue. After washing, cells were fixed with paraformaldehyde for 20 minutes, perforated by 0.5% Triton-100 for 20 minutes, blocked by 10% BSA for 30 minutes. Added Drp1 antibody and incubated overnight. Briefly, after incubated with donkey anti-rabbit and DAPI staining, the stained sections were viewed as mentioned above.
2.6 Western blot
Western blot assay was performed using standard protocols.3 mice were sacrificed after anesthetization with 20% uratan. The brains were removed rapidly, then substantia nigra was separated on ice. The tissue could be cryopreserved at -80℃ until used. High efficiency RIPA cracking fluid (R0010, Solarbio, Beijing, China) was mixed with protease inhibitor and phosphatase inhibitor (P1269, Solarbio, Beijing, China) for tissue lysis. Added 150 ul lysate to every 10 mg tissue. After 30 min, tissue lysates were obtained by centrifugation at 14,000 rpm for 10 min at 4℃. The protein concentration of the samples then was quantifified by BCA protein assay (PC0020, Solarbio, Beijing, China). After tissue lysate was mixed in loading buffer and boiled for 10 min, equivalent amounts of protein were separated on 10% SDS-polyacrylamide gel and transferred to polyvinylidene diflfluoride (PVDF) membranes. The membranes were blocked with 5% nonfat milk in TBST and incubated overnight at 4℃ with the primary antibody β-actin(1:2000), Mfn2 (1:1000), OPA1 (1:1000), Drp1 (1:1000), Nrf2 (1:1000) and HO-1 (1:1000), followed by incubation for 2 h at room temperature with goat anti-rabbit immunoglobulin (1:5000; Abcam, Cambridge, UK) and goat anti-mouse immunoglobulin (1:5000; Abcam, Cambridge, UK). The bound antibodies were then visulalized by ECL-enhanced chemilluminescence (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer's instructions. Western blot images were captured with a chemiluminescent imaging system (ThermoFisher Scientific, Massachusetts, USA). All bands were quantifified using the image system of Image J v1.51(National Institutes of Health, Bethesda, MD, USA).
For N2a cell, collections after treatment were added High efficiency RIPA to get protein. When nuclear and cytoplasmic proteins were involved, we used Nuclear Protein Extraction Kit (R0050, Beijing, Solarbio).
2.7 ROS
3 mice were sacrificed after anesthetization with 20% uratan. The brains were quickly separated and cryopreserved in liquid nitrogen. The brains were sliced with OTC embedding agent into 6 µm in the freezing microtome (Cryotome E, Thermo). Added ROS dye solution on the tissue section and incubate for 30 min away from light. After PBS washing, DAPI dye solution was used to label nucleus for 5 min away from light. All stained sections were viewed and photographed under a microscope (ZEISS Axioscope 5, Germany). The fluorescence intensity was determined using Image-pro plus 6.0 software.
For N2a cell, the Reactive Oxygen Species Assay Kit (MA0219, Dalian, China) was used following the instructions. The content of ROS was reflected by the average fluorescence intensity of DCFH-DA.
2.8 MTT
N2a cells were seeded at a density of 3×104 cells in 100µL per well for 24h for use. Cell viability were determined using MTT. The assays were performed in 96-well plates. According to previous studies and different concentrations of MPP + treatment, we used 1mM, 2mM, 4mM for 12 h as a model of oxidative stress. In order to determine the optimum concentration of Gp, cells were treated with 2.5mg/mL, 5mg/mL, 10mg/mL while MPP + co-treatment for 12 h, respectively.
Following cell treatments, 50ul of the supernatant was collected and the cell cytotoxicity was assessed according to the manufacturer’s instructions: the collected cell supernatant was incubated with the reaction mixture for 30min at room temperature, then the reaction was terminated by adding the stop solution from the kit and absorbance was measured at 490nm using a microplate reader. The absorbance values are proportional to the cytotoxicity of the cells damage. Meanwhile, 20ul of MTT (5mg/mL) was added to each well and maintained at 37℃ in a humidified incubator, after incubating for 4 h, the supernatants were removed and 100ul of DMSO was added to each well. After the formazan crystals dissolved completely, the absorbance was measured at 570 nm using a microplate reader.
2.9 Enzyme content measurement
Malondialdehyde (MDA) Content Detection Kit (BC0025, Beijing, Solarbio) was used to measure the lipid peroxidation end-product. The antioxidative enzymes SOD (K020, Wuhan, Elabscience Biotechnology) was detected using WST-1 method. The GSH (0026c, Wuhan, Elabscience Biotechnology) were detected using ELISA kits according to the manufacturer’s instructions.
3.0. Data and statistical analyses
All values are given as mean ± SEM. The significance of differences was evaluated using a parametric one-way analysis of variance (ANOVA) followed by multiple comparison test. In all cases, significance was set at p < 0.05. Statistical analyses were performed using Graphpad Prism 9.0 (Graphpad Sofware, San Diego, CA, USA).